Method of Preparing Propionic Acid-Terminated Polymers

ABSTRACT

The invention provides methods for preparing polymers bearing a terminal propionic acid. The method involves first reacting a water soluble and non-peptidic polymer comprising at least one hydroxyl group with a tertiary alkyl acrylate in the presence of a catalyst to form a propionic acid ester of the polymer, wherein the polymer has a weight average molecular weight of at least about 10,000 Da; and then treating the propionic acid ester of the polymer with a strong acid to form a propionic acid of the polymer.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 11/040,142; filed Jan. 21, 2005, which applicationclaims the benefit of priority to U.S. Provisional Patent ApplicationSer. No. 60/538,006, filed Jan. 21, 2004, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to methods for preparing water soluble andnon-peptidic polymers carrying substituted or unsubstituted propionicacid functional groups, particularly propionic acid-terminatedpoly(ethylene glycol) polymers.

BACKGROUND OF THE INVENTION

Poly(ethylene glycol) (PEG) derivatives activated with electrophilicgroups are useful for coupling to amino groups of biologically activemolecules, such as proteins. In particular, active esters of carboxylicacid derivatives of PEG have been used to attach PEG to proteins bearingamino groups.

U.S. Pat. No. 5,672,662 discloses PEG derivatives having a terminalpropionic or butanoic acid moiety that can be used to prepare activeesters suitable for conjugation to proteins or other molecules bearingamino groups. The synthetic method for propionic acid-substituted PEGdescribed in the patent involves Michael addition of poly(ethyleneglycol) to acrylonitrile followed by hydrolysis of the nitrile to formthe carboxyl group. Hydrolysis of the nitrile requires severe reactionconditions, such as treatment with concentrated sulfuric acid at 95° C.or higher. The ether linkages in PEG are sensitive to such conditionsand significant chain cleavage and reduction in yield can result fromthis process, particularly when relatively high molecular weightpolymers are involved, such as polymers having a molecular weight aboveabout 10,000 Da.

U.S. Pat. No, 5,523,479 to Sanders et al. discloses a method for formingethercarboxylic acids by reacting an alcohol having a molecular weightof 32 to 6,000 Da with a tertiary alkyl ester of an α,β-unsaturatedcarboxylic acid in the presence of a catalyst, such as potassiumhydroxide, followed by acid hydrolysis. The Sanders et al, patent doesnot address the use of higher molecular weight polymer reagents, such asPEG polymers having a molecular weight of about 10,000 Da or higher.

There is a need in the art for alternative methods for preparingpropionic acid-terminated polymers, particularly high molecular weightpolymers, in high yield without utilizing harsh reaction conditions thatcan cause chain cleavage within the polymer backbone (e.g., at theterminal methoxy group in the polymer backbone).

SUMMARY OF THE INVENTION

The present method avoids the harsh hydrolysis conditions thatcharacterize conventional methods for producing propionicacid-terminated polymers. Instead, the method of the invention firstinvolves a Michael addition reaction between a tertiary alkyl acrylateand a polymer functionalized with at least one hydroxyl group, followedby removal of a tertiary alkyl group from the terminal ester to form acarboxyl group using relatively mild conditions, such as treatment withtrifluoroacetic acid at about 50° C.

In one aspect, the present invention provides a method for preparing awater soluble and non-peptidic polymer functionalized with at least onepropionic acid group, the method comprising:

-   -   i) reacting a water soluble and non-peptidic polymer comprising        at least one hydroxyl group with a tertiary alkyl acrylate or        substituted tertiary alkyl acrylate in the presence of a        catalyst to form a substituted or unsubstituted propionic acid        ester of the polymer, wherein the polymer has a weight (or        number) average molecular weight of at least about 10,000 Da;        and    -   ii) treating the substituted or unsubstituted propionic acid        ester of the polymer with a strong acid, such as (for example)        trifluoroacetic acid, trifluoromethanesulfonic acid, formic        acid, hydrochloric acid, or p-toluenesulfonic acid, to form a        propionic or substituted propionic acid of the polymer.

The tertiary alkyl acrylate can be α- or β-substituted and exemplarysubstituting groups include halo, hydroxyl, thiol, alkylthio, acyl,acyloxy, nitro, cyano, azido, trihalomethyl, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl, andsubstituted heteroaryl. In one preferred embodiment, the tertiary alkylacrylate is α- or β-substituted, preferably α-substituted, with methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, orbenzyl. Preferred tertiary alkyl groups of the tertiary alkyl acrylateinclude tert-butyl, tert-amyl, α,α′-dimethylbenzyl, trityl, 1-adamantyl,and 2-methyl-2-adamantyl.

In one or more embodiments, the tertiary alkyl acrylate has thestructure:

wherein:

-   -   R₁ and R₂ are each independently selected from the group        consisting of hydrogen, halo, hydroxyl, thiol, alkylthio, acyl,        acyloxy, nitro, cyano, azido, trihalomethyl, alkyl, substituted        alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted        alkoxy, aryl, substituted aryl, heterocycle, substituted        heterocycle, heteroaryl, and substituted heteroaryl; and    -   R₃—R₅ (that is, each of R₃, R₄ and R₅) are each independently        alkyl, substituted alkyl, aryl or substituted aryl.

Preferably, R₃, R₄, and R₅, are each methyl, ethyl, or phenyl, and R₁and R₂ are hydrogen or R₁ is hydrogen and R₂ is methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl.

The catalyst used in the Michael addition reaction is preferably aquaternary ammonium hydroxide, such as a tetraalkyl ammonium halide orhydroxide (e.g., tetramethyl ammonium hydroxide, tetraethyl ammoniumhydroxide, tetrapropyl ammonium hydroxide, or tetrabutyl ammoniumhydroxide, as well as the corresponding halides). In one or moreembodiments, the quaternary ammonium hydroxide has the structure:

wherein each R is independently alkyl or substituted alkyl (e.g., C1-C8alkyl). In addition, ⁻OH counter ion can be substituted for halo,wherein halo represents fluoro, chloro, bromo, and iodo.

Both the reacting step i) and treating step ii) can be conducted in thepresence of an organic solvent, such as dichloromethane (DCM),tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide(DMSO), acetonitrile, toluene, benzene, xylene, phenylacetonitrile,nitrobenzene, tetrachloroethylene, anisole, chlorobenzene, andtert-butanol.

The polymer bearing at least one hydroxyl group is preferablypoly(ethylene glycol), but can be any other water-soluble andnon-peptidic polymer, such as other poly(alkylene glycols),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxyacetic acid), poly(acrylic acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines,poly(N-acryloylmorpholine), and copolymers or terpolymers thereof. Thepolymer preferably has a weight (or number) average molecular weight ofabout 10,000 to about 100,000 Da, more preferably about 10,000 to about40,000 Da. The PEG polymer or other polymer can have any of a variety ofstructures and geometric configurations, including, for example,monofunctional PEG, difunctional PEG, and branched PEG.

Following formation of the carboxylic acid group, the propionic acidfunctionalized polymer can be derivatized to form an acid derivativesuch as acyl halide, acyl pseudohalide, ester, anhydride, amide, imide,or hydrazide. Furthermore, the acid or certain functionalized polymers,e.g. active esters, can be used as intermediates to react withappropriate reagents or other small molecules or short polymeric speciesto form yet additional reactive derivatives such as maleimides, thiols,reactive disulfides, acetals, aldehydes and the like. In one embodiment,the propionic acid-functionalized polymer is derivatized to form anactive ester. Exemplary active ester groups includeN-hydroxysuccinimidyl ester, o-, m-, or p-nitrophenyl ester,1-hydroxybenzotriazolyl ester, imidazolyl ester, andN-hydroxysulfosuccinimidyl ester.

In a preferred embodiment of the invention, the method for preparing apoly(ethylene glycol) (PEG) polymer functionalized with at least onepropionic acid group comprises:

-   -   i) reacting a PEG polymer with a tertiary alkyl acrylate or        substituted tertiary alkyl acrylate in the presence of a        quaternary ammonium hydroxide to form a propionic acid or        substituted propionic acid ester of PEG, wherein the PEG polymer        is a monofunctional PEG, difunctional PEG, or branched PEG        molecule comprising 1 to about 25 hydroxyl groups and having a        number average molecular weight of at least about 10,000 Da;    -   ii) treating the propionic acid or substituted propionic acid        ester of PEG with a strong acid, such as (for example)        trifluoroacetic acid, trifluoromethanesulfonic acid, formic        acid, hydrochloric acid, or p-toluenesulfonic acid, to form a        PEG polymer functionalized with at least one propionic acid or        substituted propionic acid group;    -   iii) optionally, chromatographically purifying the PEG polymer        functionalized with at least one propionic acid group;    -   iii) optionally, derivatizing the propionic acid or substituted        propionic acid functionalized PEG to form, for example, an        active ester selected from the group consisting of        N-hydroxysuccinimidyl ester, o-, m-, or p-nitrophenyl ester, 1-        hydroxybenzotriazolyl ester, imidazolyl ester, and        N-hydroxysulfosuccinimidyl ester; and    -   v) optionally, chromatographically purifying the active ester of        PEG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification, the singular fowls“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—O(CH₂CH₂O)_(m)—” or “—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂—,” where (m) is 3 to3000, and the terminal groups and architecture of the overall PEG mayvary. “PEG” means a polymer that contains a majority, that is to say,greater than 50%, of subunits that are —CH₂CH₂O—. One commonly employedPEG is end-capped PEG. When PEG is defined as “—O(CH₂CH₂O)_(m)—” the endcapping group is generally a carbon-containing group typically comprisedof 1-20 carbons and is preferably alkyl (e.g., methyl, ethyl or benzyl)although saturated and unsaturated forms thereof, as well as aryl,hetcroaryl, cyclo, heterocyclo, and substituted forms of any of theforegoing are also envisioned. When PEG is defined as“—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂—,” the end capping group is generally acarbon-containing group typically comprised of 1-20 carbon atoms and anoxygen atom that is covalently bonded to the group and is available forcovalently bonding to one terminus of the PEG. In this case, the groupis typically alkoxy (e.g., methoxy, ethoxy or benzyloxy) and withrespect to the carbon-containing group can optionally be saturated andunsaturated, as well as aryl, heteroaryl, cyclo, heterocyclo, andsubstituted forms of any of the foregoing. The other (“non-end-capped”)terminus is a typically hydroxyl, amine or an activated group that canbe subjected to further chemical modification when PEG is defined as“—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂-.” In addition, the end-capping group canalso be a silane. Specific PEG forms for use in the invention includePEGs having a variety of molecular weights, structures or geometries(e.g., branched, linear, forked PEGs, multifunctional, and the like), tobe described in greater detail below.

The end-capping group can also advantageously comprise a detectablelabel. When the polymer has an end-capping group comprising a detectablelabel, the amount or location of the polymer and/or the moiety (e.g.,active agent) to which the polymer is attached can be determined byusing a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, and thelike.

“Molecular mass” in the context of a water-soluble, non-peptidic polymerof the invention such as PEG, refers to the weight average molecularweight of a polymer, typically determined by size exclusionchromatography, light scattering techniques, or intrinsic viscositydetermination in an organic solvent like 1,2,4-trichlorobenzene. Thepolymers of the invention are typically polydisperse, possessing lowpolydispersity values of less than about 1.05.

“Activated carboxylic acid” means a functional derivative of acarboxylic acid that is more reactive than the parent carboxylic acid,in particular, with respect to nucleophilic acyl substitution. Activatedcarboxylic acids include but are not limited to acid halides (such asacid chlorides), anhydrides, amides and esters.

The term “reactive” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e, a “nonreactive” or “inert” group).

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive group beingprotected as well as the reaction conditions to be employed and thepresence of additional reactive or protecting groups in the molecule, ifany. Protecting groups known in the art can be found in Greene, T. W.,et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley &Sons, New York, N.Y. (1999).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof

The term “spacer” or “spacer moiety” is used herein to refer to an atomor a collection of atoms optionally used to link interconnectingmoieties such as a terminus of a water-soluble polymer portion and anelectrophile. The spacer moieties of the invention may be hydrolyticallystable or may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includeethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-methylpropyl (isobutyl),3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl or cycloalkylene when three or more carbon atoms arereferenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, such as ethynyl, n-propynyl, isopentynyl, n-butynyl,octynyl, decynyl, and so forth.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C1-C20 alkyl (e.g., methyl, ethyl, propyl, benzyl,etc.), preferably C1-C8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C3-C8cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy; phenyl; substitutedphenyl; and the like.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Substituted aryl” is aryl having one or more non-interfering groups asa substituent. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta, or para).

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Electrophile” refers to an ion or atom or a neutral or ionic collectionof atoms having an electrophilic center, i.e., a center that is electronseeking or capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or a neutral or ionic collectionof atoms having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or capable of reacting with an electrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include, butare not limited to, carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups contained therein, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically contain from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within thepolymer backbone.

A “difunctional” polymer means a polymer having two functional groupscontained therein, either the same (i.e., homodifunctional) or different(i.e., heterodifunctional).

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

Unless otherwise noted, molecular weight is expressed herein as numberaverage molecular weight (M_(n)), which is defined as

$\frac{\sum\; {NiMi}}{\sum\; {Ni}},$

wherein Ni is the number of polymer molecules (or the number of moles ofthose molecules) having molecular weight Mi,

As used herein, “non-peptide” refers to a polymer backbone substantiallyfree of peptide linkages. However, the polymer backbone may include aminor number of peptide linkages spaced along the length of thebackbone, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

II. Method of Preparing Propionic Acid Functionalized Polymers

The method of the invention provides a synthetic route for forming watersoluble and non-peptidic polymers functionalized with at least onepropionic acid group. The method involves reaction of a polymercomprising at least one hydroxyl group, such as monofunctional,difunctional or multifunctional PEG molecules, with a tertiary alkylacrylate reagent in a Michael addition reaction, which results in apolymer substituted with at least one tertiary alkyl ester of propionicacid. The ester is then hydrolyzed under relatively mild conditions ascompared to the hydrolysis conditions required for a nitrile group. Thehydrolysis conditions used in the method of the invention do not causeyield-reducing degradation and chain cleavage of the polymer backbone,thereby making the method particularly well-suited for higher molecularweight polymers, such as polymers having a molecular weight of greaterthan about 10,000 Da.

In a preferred embodiment, a catalyst is used to promote the Michaeladdition reaction. The choice of catalyst is particularly important forhigher molecular weight polymer starting materials because, as indicatedin Comparative Example 1, certain catalysts in the art are unable toadvance the Michael addition reaction to any significant degree. Apreferred catalyst comprises a quaternary ammonium hydroxide. Exemplaryquaternary ammonium hydroxides include tetramethyl ammonium hydroxide,tetraethyl ammonium hydroxide, tetrapropyl ammonium hydroxide, andtetrabutyl ammonium hydroxide. Quaternary ammonium hydroxides can beused directly or generated in situ from corresponding quaternaryammonium salts, preferably halides. If a quaternary ammonium halide saltis used, it is necessary to activate the ammonium salt by addition of analkali metal or alkaline earth metal hydroxide, such as KOH or NaOH, asshown in Examples 1 and 3. The catalyst can be dissolved in the sameorganic solvent as the reaction reagents or added in the form of anaqueous solution.

In one embodiment, the quaternary ammonium hydroxide has the structure:

wherein each R is independently alkyl or substituted alkyl, preferablysubstituted or unsubstituted C1-8 alkyl.

Hydrolysis of the tertiary alkyl ester group can be accomplished bytreatment with any strong acid, such as various solutions of mineralacids (e.g., hydrohalic acids, sulfuric acid, phosphorous acid, and thelike) or organic acids. One preferred acid is trifluoroacetic acid(TFA). Examples of other suitable acids include formic acid,hydrochloric acid, p-toluenesulfonic acid, and trifluoromethanesulfonicacid.

The reagents in both the Michael addition step and the subsequenthydrolysis step are preferably dissolved in a suitable organic solvent.Exemplary organic solvents include dichloromethane (DCM),tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide(DMSO), acetonitrile, toluene, benzene, xylene, phenylacetonitrile,nitrobenzene, tetrachloroethylene, anisole, chlorobenzene, tert-butanol,and the like.

The Michael addition reaction can be carried out at any temperature thatmaintains the polymer starting material (i.e., the polymeric alcohol) ina liquid state. Preferably, the temperature is about 20 to about 120°C., more preferably about 20 to about 60° C. Such temperatures are lowerthan those typically suggested, and represents relatively gentlerreaction conditions, especially in the presence of basic catalyst,thereby practically eliminating degradation and chain cleavage of thepolymer backbone. The Michael addition reaction time is typically about6 hours to about 24 hours. The polymer is typically reacted with thetertiary alkyl acrylate using an excess of the acrylate reagent (e.g.,up to about 30 fold molar excess) in order to promote substantiallycomplete conversion of the polymeric alcohol. The catalyst is typicallypresent in an amount of about 0.05 weight percent to about 20 weightpercent based on the weight of the starting polymer.

The acid-promoted hydrolysis step typically comprises treating thepropionic acid ester of the polymer with a strong acid, preferably anorganic acid, at a temperature of about 20 to about 100° C., preferablyat the lower end of the range, for about 0.5 hours to about 6 hours. Theuse of organic acids with relatively lower temperatures representsmilder reaction conditions than required for the hydrolysis of, forexample, nitriles as evidenced in U.S. Pat. No. 5,672,662, with theresult of practically eliminating degradation and chain cleavage of thepolymer backbone. Following conversion of the ester to the desired acid,any organic solvents or acids, such as trifluoroacetic acid, can beremoved by distillation. Thereafter, the desired product is preferablydissolved in deionized water and treated with a strong base to hydrolyzeany esters of residual polymeric alcohol (e.g., PEG-OH) and acid (e.g.,trifluoroacetic acid), followed by treatment with a strong mineral acid(for pH adjustment) to convert the resulting salt of thepolymer-propionic acid (e.g., PEG-propionic acid) to the free acid form.The product is then extracted using a chlorinated solvent such asdichloromethane and concentrated. The desired product can then bepurified using methods known in the art for polymers of this type.

Using the method of the invention, propionic acid-functionalizedpolymers can be produced in high yield with a high degree of polymersubstitution. Typically, the percentage of polymer substitution of thepropionic acid ester onto the polymeric alcohol is at least about 70%,preferably at least about 80%, and most preferably at least about 90%substitution. The product yield is typically at least about 60%, morepreferably at least about 70%, and most preferably at least about 80%.

A general reaction scheme of the present invention, denoted as ReactionScheme I, is shown below. As shown, an mPEG-OH molecule is reacted witha tert-butyl acrylate substituted at the α-carbon as described ingreater detail below. A quaternary ammonium halide activated with KOH isused as a catalyst for the Michael addition step. Trifluoroacetic acid(“TFA”) is used in an acid-promoted hydrolysis step to remove thetert-butyl protecting group. In Reaction Scheme I, “Me” representsmethyl, “n” represents the number of repeating ethylene oxide monomers,“t-Bu” represents t-butyl, and R₂ is as defined in Section II.B.

In exemplary Reaction Scheme II below, a method according to theinvention is outlined that includes formation of an active NHS esterfollowing purification of the propionic acid functionalized polymer.

As explained more fully below, the polymeric alcohol starting materialmay comprise any water soluble and non-peptidic polymer having any of awide variety of geometric configurations (e.g., linear, branched,forked, and so forth). For the sake of simplicity, the above reactionschemes illustrate use of a monofunctional polymer having a singlehydroxyl group.

However, the polymer may comprise more than one hydroxyl group, such as1 to about 25 hydroxyl groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore hydroxyl groups).

If a methoxy-PEG-OH is used as the raw material, as shown in ReactionScheme II, this process produces the active ester with the polymerbackbone chain impact. That is, there is no observable chain cleavagefrom the acid-catalyzed hydrolysis of the ester. While not wishing to bebound by theory, it is believed the absence of chain cleavage is theresult of using relatively reduced temperatures and a milder organicacid such as trifluoroacetic acid followed by base treatment. This lackof chain cleavage and especially the lack of terminal demethylation(thereby resulting in a lack of demethylated product), results in asignificant difference and advantage over the propionic acid-terminatedpolymers (and corresponding active esters) prepared in accordance withthe process described in the U.S. Pat. No. 5,672,662. Additionally, theconjugates prepared from active reagents derived from PEG-propionic acidmanufactured using U.S. Pat. No. 5,672,662 would suffer by comparison toconjugates prepared using the present method.

A. Water Soluble and Non-Peptidic Polymers

The polymer should be non-toxic and biocompatible, meaning that thepolymer is capable of coexistence with living tissues or organismswithout causing harm. When referring to the polymer, it is to beunderstood that the polymer can be any of a number of water soluble andnon-peptidic polymers, such as those described herein as suitable foruse in the present invention. Preferably, poly/ethylene glycol) (i.e.,PEG) is the polymer. The term PEG includes poly(ethylene glycol) in anyof a number of geometries or forms, including linear forms (e.g.,methoxy-PEG-OH, benzyloxy-PEG-OH, or HO-PEG-OH), branched or multi-armforms (e.g., forked PEG or PEG attached to a polyol core), pendant PEG,or PEG with degradable linkages therein, to be more fully describedbelow.

The polymer comprises at least one hydroxyl group capable of reactingwith a tertiary alkyl acrylate reagent in a Michael addition reaction.In addition to the one or more hydroxyl groups, the polymer may compriseother functional groups that would not interfere with the Michaeladdition reaction, such as acetal of an aldehyde having a carbon lengthof 1 to 25 carbons (e.g., acetaldehyde, propionaldehyde, andbutyraldehyde), alkenyl, acrylate, methacrylate, acrylamide, activesulfone, hydrazide, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridinc, iodoacetamide, epoxide, glyoxal, biotin,dione, mesylate, tosylate, and tresylate.

The number of hydroxyl groups carried by the polymer and the position ofthe functional groups may vary. Typically, the polymer will comprise 1to about 25 hydroxyl groups, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10hydroxyl groups. Linear polymers, such as PEG polymers, will typicallycomprise one or two hydroxyl groups positioned at the terminus of thepolymer chain. If the PEG polymer is monofunctional (i.e., mPEG), thepolymer will include a single hydroxyl group. If the PEG polymer isdifunctional, the polymer may contain two hydroxyl groups, one at eachterminus of the polymer chain, or may contain a single hydroxyl groupand a different functional group at the opposing terminus. As would beunderstood, multi-arm or branched polymers may comprise a greater numberof hydroxyl groups.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. Generally speaking, amulti-armed or branched polymer possesses two or more polymer “arms”extending from a central branch point. For example, an exemplarybranched PEG polymer has the structure:

wherein PEG₁ and PEG₂ are PEG polymers in any of the forms or geometriesdescribed herein, and which can be the same or different, and L′ is ahydrolytically stable linkage. An exemplary branched PEG of Formula IIIhas the structure:

wherein poly_(a) and poly_(b) are PEG backbones, such as methoxypoly(ethylene glycol); R″ is a nonreactive moiety, such as H, methyl ora PEG backbone; and P and Q are nonreactive linkages. In a preferredembodiment, the branched PEG polymer is methoxy poly(ethylene glycol)disubstituted lysine.

The branched PEG structure of Formula IV can be attached to a thirdoligomer or polymer chain as shown below:

wherein PEG₃ is a third PEG oligomer or polymer chain, which can be thesame or different from PEG₁ and PEG₂.

In another multi-arm embodiment, the polymer comprises a central coremolecule derived from a polyol or polyamine, the central core moleculeproviding a plurality of attachments sites suitable for covalentlyattaching polymer arms to the core molecule in order to form a multi-armpolymer structure. An exemplary multi-arm polymer of this type has thestructure:

R(-L″-PEG-OH)_(q)  (Formula VI)

wherein:

R is the hydrocarbon chain of the polyol or polyamine core molecule,typically comprising about 3 to about 150 carbon atoms, preferably about3 to about 50 carbon atoms, and most preferably about 3 to about 10carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10), optionally substitutedwith one or more heteroatoms (e.g., O, S, or N) in the hydrocarbonchain, and which may be linear or cyclic;

L″ is a linkage formed from reaction of the polyol or polyamine coremolecule with the polymer arms (e.g., —O— or —NH—C(O)—), and can serveas a spacer moiety;

PEG is a poly(ethylene glycol) polymer segment; and

q is an integer from 3 to about 25, more preferably to about 10, mostpreferably 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8).

The central core molecule in the multi-arm embodiment described above isderived from a molecule that provides a number of polymer attachmentsites equal to the desired number of water soluble and non-peptidicpolymer arms. Preferably, the central core molecule of the multi-armpolymer structure is the residue of a polyol or a polyamine bearing atleast three hydroxyl or amino groups available for polymer attachment. A“polyol” is a molecule comprising a plurality of available hydroxylgroups. A “polyamine” is a molecule comprising a plurality of availableamino groups. Depending on the desired number of polymer arms, thepolyol or polyamine will typically comprise 3 to about 25 hydroxyl oramino groups, preferably 3 to about 10, most preferably 3 to about 8(e.g., 3, 4, 5, 6, 7, or 8). The polyol or polyamine may include otherprotected or unprotected functional groups as well without departingfrom the invention. Although the spacing between hydroxyl or aminogroups will vary, there is typically 1 to about 20 atoms, such as carbonatoms, between each hydroxyl or amino group, preferably 1 to about 5.The particular polyol or polyamine chosen will depend on the desirednumber of hydroxyl or amino groups needed for attachment to the polymerarms.

The polyol or polyamine core will typically have the structureR—(OH)_(p) or R—(NH₂)_(p) prior to reaction with the polymer arms,wherein R is a hydrocarbon chain, typically comprising about 3 to about150 carbon atoms, preferably about 3 to about 50 carbon atoms, and mostpreferably about 3 to about 10 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9,or 10), optionally substituted with one or more heteroatoms (e.g., 0, S,or N) in the hydrocarbon chain, and which may be linear or cyclic, and pis the number of hydroxyl or amino groups and is typically 3 to about25, preferably 3 to about 10, more preferably 3 to about 8 (e.g., 3, 4,5, 6, 7, or 8).

Polyols that are suitable for use as the polymer core are nearlylimitless. Aliphatic polyols having from 1 to about 10 carbon atoms andfrom 1 to about 10 hydroxyl groups may be used, including ethyleneglycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkylcycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.More examples of aliphatic polyols include derivatives ofglyceraldehyde, glucose, ribose, mannose, galactose, and relatedstereoisomers. Aromatic polyols may also be used, such as1,1,1-tris(4′-hydroxyphenyl) alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,2,6-bis(hydroxyalkyl)cresols,2,2′alkylene-bis(6-t-butyl-4-alkylphenols),2,2′-alkylene-bis(t-butylphenols), catechol, alkylcatechols, pyrogallol,fluoroglycinol, 1,2,4-benzenetriol, resorcinol, alkylresorcinols,dialkylresorcinols, orcinol monohydrate, olivetol, hydroquinone,alkylhydroquinones, 1,1-bi-2-naphthol, phenyl hydroquinones,dihydroxynaphthalenes, 4,4′-(9-fluorenylidene)-diphenol, anthrarobin,dithranol, bis (hydroxyphenyl) methane biphenols, dialkyistilbesterols,bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,meso-hexesterol, nordihydroguaiaretic acid, calixarenes and derivativesthereof, tannic acid, and the like. Other core polyols that may be usedinclude crown ether, cyclodextrins, dextrins and other carbohydrates(e.g., monosaccharides, oligosaccharides, and polysaccharides, starchesand amylase).

Preferred polyols include glycerol, sugars such as sorbitol orpentaerythritol, and glycerol oligomers, such as hexaglycerol. A 21-armpolymer can be synthesized using hydroxypropyl-β-cyclodextrin, which has21 available hydroxyl groups.

Exemplary polyamines include aliphatic polyamines such as diethylenetriamine, N,N′,N″-trimethyldiethylene triamine, pentamethyl diethylenetriamine, triethylene tetramine, tetraethylene pentamine, pentaethylenehexamine, dipropylene triamine, tripropylene tetramine,bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, andN,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines thatcan be used in the present invention include putrescine, spermidine, andspermine. Numerous suitable pentamines, tetramines, oligoamines, andpentamidine analogs suitable for use in the present invention aredescribed in Bacchi et al., Antimicrobial Agents and Chemotherapy,January 2002, p. 55-61, Vol. 46, No. 1, which is incorporated byreference herein.

The PEG polymer may alternatively comprise a forked PEG. Generallyspeaking, a polymer having a forked structure is characterized as havinga polymer chain attached to two or more functional groups via covalentlinkages extending from a hydrolytically stable branch point in thepolymer. An example of a forked PEG is represented by PEG-YCH(-L-Z)₂,where Y is a linking group and Z is an activated terminal group forcovalent attachment to a biologically active agent. The Z group islinked to CH by a linker, L, which is a chain of atoms of definedlength. U.S. Pat. No. 6,362,254, the contents of which are incorporatedby reference herein, discloses various forked PEG structures capable ofuse in the present invention. The chain of atoms, L, linking the Zfunctional groups (e.g., hydroxyl groups) to the branching carbon atomserve as a tethering group and may comprise, for example, an alkylchain, ether linkage, ester linkage, amide linkage, or combinationsthereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups (e.g., hydroxyl groups) covalently attached along the length ofthe PEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylenc group.

Although less preferred, in addition to the above-described forms ofPEG, the polymer can also be prepared with an enzymatically degradablelinkage or one or more hydrolytically stable or degradable linkages inthe polymer backbone, including any of the above described polymers. Forexample, PEG can be prepared with ester linkages in the polymer backbonethat are subject to hydrolysis. As shown below, this hydrolysis resultsin cleavage of the polymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between acid derivatives and an alcohol; andoligonucleotide linkages formed by, for example, a phosphoramiditegroup, e.g., at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG. Any ofa variety of other polymers comprising other non-peptidic and watersoluble polymer chains can also be used in the present invention. Thepolymer can be linear, or can be in any of the above-described forms(e.g., branched, forked, and the like). Examples of suitable polymersinclude, but are not limited to, other poly(alkylene glycols),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazolines, poly(N-acryloylmorpholine), such as described in U.S.Pat. No. 5,629,384, which is incorporated by reference herein in itsentirety, and copolymers, terpolymers, and mixtures thereof.

Different polymers can be incorporated into the same polymer backbone.For example, one or more of the PEG molecules in the branched structuresshown in Formulas III-VI can be replaced with a different polymer type.Any combination of water soluble and non-peptidic polymers isencompassed within the present invention.

The molecular weight of the polymer will vary depending on the desiredapplication, the configuration of the polymer structure, the degree ofbranching, and the like. Generally, polymers having a molecular weightof about 10,000 Da to about 100,000 Da are useful in the presentinvention, preferably about 10,000 Da to about 60,000 Da, and morepreferably about 10,000 Da to about 40,000 Da. Exemplary polymerembodiments have a molecular weight of approximately 10,000 Da, 15,000Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, and 40,000 Da. However,lower molecular weight polymers can also be used without departing fromthe present invention, such as polymers having a molecular weight as lowas about 100 Da (e.g., polymers having a molecular weight of about 250Da, about 500 Da, about 750 Da, about 1,000 Da, about 1,500 Da, about2,500 Da, and about 5,000 Da).

Useful exemplary weight average molecular weights of the polymersinclude about 100 Da, about 200 Da, about 300 Da, about 400 Da, about500 Da, about 600 Da, about 700 Da, about 750 Da, about 800 Da, about900 Da, about 1,000 Da, about 2,000 Da, about 2,500 Da, about 3,000 Da,about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about7,500 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 11,000Da, about 12,000 Da, about 12,500 Da, about 15,000 Da, about 20,000 Da,about 25,000 Da, and 30,000 Da, about 40,000 Da, about 50,000 Da, about60,000 Da, about 70,000 Da, about 75,000 Da, about 80,000 Da, about85,000 Da, about 90,000 Da, about 100,000 Da, and about 120,000 Da.

With respect to singly branched versions of the polymer, exemplaryranges of useful sizes for the total molecular weight of the polymer (asbased essentially on the combined weights of the two water solublepolymer portions) include the following: from about 200 Da to about100,000 Da; from about 1,000 Da to about 80,000 Da; from about 2,000 Dato about 60,000 Da; from about 4,000 Daltons to about 50,000 Daltons;and from about 10,000 Da to about 40,000 Da. More particularly, totalweight average molecular weight of a singly branched version of thepolymer of the invention corresponds to one of the following: about 400;about 1,000; about 1,500; about 2,000; about 3000; about 4,000; about10,000; about 15,000; about 20,000; about 30,000; about 40,000; about50,000; about 60,000; or about 80,000.

With respect to PEG, wherein a structure comprising a repeating ethyleneoxide monomer, such as “—(CH₂CH₂O)_(m)—” or “—(OCH₂CH₂)_(m)—” [as in,for example, H₃CO—(CH₂CH₂O)_(m)—CHR₂—CHR₂—C(O)—Y, where R₁ R₂ and Y areas defined for Formula VIII] exemplary values for m include: from about3 to about 3,000; from about 10 to about 3,000; from about 15 to about3,000; from about 20 to about 3,000; from about 25 to about 3,000; fromabout 30 to about 3,000; from about 40 to about 3,000; from about 50 toabout 3,000; from about 55 to about 3,000; from about 75 to about 3,000;from about 100 to about 3,000; and from about 225 to about 3,000.

B. Tertiary Alkyl Acrylate Reagent

The tertiary alkyl acrylate can be α- or β-substituted. Exemplarysubstituting groups include halo, alkylthio, acyl, acyloxy, nitro,cyano, azido, trihalomethyl, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substitutedaryl, heterocycle, substituted heterocycle, heteroaryl, and substitutedheteroaryl. The tertiary alkyl acrylate is preferably unsubstituted atthe β carbon. The acrylate reagent is advantageously substituted at thea carbon with an alkyl or aryl group that provides steric hindrance tothe final carboxylic acid group. As taught in U.S. Pat. No. 6,495,659,which is incorporated herein by reference in its entirety, the stericeffects of a side chain attached to the a carbon can favorably affectthe hydrolytic stability of drug conjugates formed using the polymeracid. In one preferred embodiment, the tertiary alkyl acrylate is α- orβ-substituted, preferably α-substituted, with methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl.

In one embodiment, the tertiary alkyl acrylate has the structure:

wherein:

-   -   R₁ and R₂ are each independently selected from the group        consisting of hydrogen, halo, alkylthio, acyl, acyloxy, nitro,        cyano, azido, trihalomethyl, alkyl, substituted alkyl,        cycloalkyl, substituted cycloalkyl, alkoxy, aryl, substituted        aryl, heterocycle, substituted heterocycle, heteroaryl, and        substituted heteroaryl; and    -   R₃-R₅ are each independently alkyl, substituted alkyl, aryl or        substituted aryl.

Preferably, R₃, R₄, and R₅, are each methyl, ethyl, or phenyl, and R₁and R₂ are hydrogen or R₁ is hydrogen and R₂ is methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl. In onepreferred embodiment, the tertiary alkyl group is tert-butyl (i.e., eachof R₃-R₅ is methyl). Other exemplary tertiary alkyl groups includetert-amyl, α,α″-dimethylbenzyl, trityl, 1-adamantyl, and2-methyl-2-adamantyl.

Preferred tertiary alkyl acrylates include tert-butyl acrylate andtert-butyl methacrylate, which are commercially available fromSigma-Aldrich Corporation, St. Louis, Mo. Other exemplary tertiary alkylacrylates include tert-butyl esters of crotonic acid or isocrotonicacid. Additionally, other tertiary alkyl or tertiary cycloalkyl acrylateor methacrylates are suitable for use in the present invention.

C. The Polymer Bearing at Least One Propionic Acid Group

Following the method described herein, the water soluble andnon-peptidic polymer will bear at least one terminal propionic acidgroup. An exemplary polymer will correspond to the following structure:

POLY-CHR₁—CHR₂—COOH  (Formula VII)

wherein POLY is the residue of a water soluble and non-peptidic polymer(such as PEG), and R₁ and R₂ (as discussed above in Section II.B) areeach independently selected from the group consisting of hydrogen, halo,alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl, andsubstituted heteroaryl. In terms of POLY, the corresponding watersoluble and non-peptidic polymers are discussed above in Section II.A,PEG [e.g., “—(CH₂CH₂O)_(m)—” or “—(OCH₂CH₂)_(m)—”] is a particularlypreferred POLY and is discussed above in Section II.A. In one or moreembodiments, branched versions of the polymer are preferred.

If desired, the propionic acid functionalized-polymer can be furthermodified to form useful reactive derivatives of carboxylic acids usingmethodology known in the art. Thus, the invention includes polymersobtainable from and/or obtained from the described propionicacid-functionalized polymers. For example, the carboxylic acid can befurther derivatized to form acyl halides, acyl pseudohalides, such asacyl cyanide, acyl isocyanate, and acyl azide, neutral salts, such asalkali metal or alkaline-earth metal salts (e.g. calcium, sodium, orbarium salts), esters, anhydrides, amides, imides, hydrazides, and thelike. In addition, the carboxylic acid can be reduced to form analdehyde, either directly from the carboxylic acid using a suitablereducing agent, or indirectly through an amide, nitrile or ester using asuitable reducing agent. Also, the acid or certain functionalizedpolymers, e.g. active esters, may be used as intermediates to react withappropriate reagents or other small molecules or short polymeric speciesto form yet additional reactive derivatives such as maleimides, thiols,reactive disulfides, acetals, aldehydes and the like.

In a preferred embodiment, the propionic acid is esterified to form anactive ester, such as an N-hydroxysuccinimidyl ester, o-, m-, orp-nitrophenyl ester, 1-hydroxybenzotriazolyl ester, imidazolyl ester, orN-hydroxysulfosuccinimidyl ester. The propionic acid or reactivederivative thereof attached to the polymer preferably has the structure:

—CHR₁—CHR₂—C(O)—Y  (Formula VIII)

wherein R₁ and R₂ (as discussed above in Section II.B) are eachindependently selected from the group consisting of hydrogen, halo,alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl, andsubstituted heteroaryl, and Y is selected from the group consisting ofhydrogen, halo, hydroxy, amino, substituted amino, —NCO, —NCS, N₃, —CN,and —O—R′, wherein R′ is N-succinimidyl, nitrophenyl, benzotriazolyl,imidazolyl, N-sulfosuccinimidyl, N-phthalimidyl, N-glutarimidyl,N-tetrahydrophthalimidyl, N-norbornene-2,3-dicarboximidyl, andhydroxy-7-azabenzotriazolyl.

In one embodiment, Y is a substituted amino having the structure —NHR₆,wherein R₆ is any organic group that may contain additional reactivefunctional groups (e.g., aldehyde, maleimide, mercapto, and the like)and where the additional functional group or groups are separated fromthe carbonyl carbon by an alkylene chain (e.g., C1-6 alkylene chain)and, optionally, an additional linker, such as a short PEG chain andanother alkylene chain (e.g., alkylene-PEG-alkylene).

Exemplary polymers that can be prepared through the described propionicacid-functionalized polymers include the following:

wherein m is defined as in Section II.A, above, and EC is a residue of amoiety selected from the group consisting of fluorescein, biotin,acrylate, vinylsulfone, maleimide, tert-butyl carbonyl (t-Boc), and9-fluorenylmethoxycarbonyl (Fmoc).

In some situations, it is preferred that the polymer bearing a propionicacid is not methoxy PEG propionic acid having a weight average molecularweight of about 20,000 Da [i.e., H₃CO—(CH₂CH₂O)_(m)—CH₂—CH₂—COOH,wherein m does not result in a weight average molecular weight of about20,000 Da], and not methoxy PEG propionic acid having a weight averagemolecular weight of about 30,000 Da [i.e.,H₃CO—(CH₂CH₂O)_(m)—CH₂—CH₂—COOH, wherein m does not result in a weightaverage molecular weight of about 30,000 Da]. Furthermore, in somesituations, it is preferred that the polymer bearing a propionic acid isnot methoxy PEG (or other polymer) propionic acid having a weightaverage molecular weight of between about 17,500 Da and 22,500 Da, andnot methoxy PEG propionic acid having a weight average molecular weightof between about 27,500 Da and 32,500 Da. In still other situations, itis preferred that the weight average molecular weight of the methoxy PEGpropionic acid is greater than 35,000 Da. Preferred polymers bearing apropionic acid comprise branched (i.e., singly branched ormulti-branched) structures as previously discussed.

D. Biologically Active Molecules for Conjugation

The propionic acid-terminated polymer produced by the method of theinvention, or a reactive derivative thereof, can be used to formconjugates with biologically active molecules, particularly biologicallyactive molecules carrying nucleophilic functional groups, such as aminogroups. Such polymer conjugates can be formed using known techniques forcovalent attachment of an activated polymer, such as an activated PEG,to a biologically active agent (See, for example, POLY(ETHYLENE GLYCOL)CHEMISTRY AND BIOLOGICAL APPLICATIONS, American Chemical Society,Washington, DC (1997)).

With respect to polymers used in conjugation,electrophilically-activated polymer derivatives, such as active esters,are useful for conjugation to amino groups on proteins or otherbiologically active molecules. Conjugation of a polymer bearing anactive carboxylic acid ester with an amino group on a biologicallyactive molecule results in formation of a stable amide bond between thepolymer and the biologically active molecule.

A biologically active agent for use in coupling to polymer formed by themethod of the invention may be any one or more of the following.Suitable agents may be selected from, for example, hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagnonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Examples of active agents suitable for use in covalent attachment to apolymer prepared by the method of the invention include, but are notlimited to, calcitonin, erythropoietin (EPO), Factor VIII, Factor IX,ceredase, cerezyme, cyclosporin, granulocyte colony stimulating factor(GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (HGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hoimone (FSH), insulin-like growthfactor (IGF), insulintropin, macrophage colony stimulating factor(M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocytegrowth factor (KGF), glial growth factor (GGF), tumor necrosis factor(TNF), endothelial growth factors, parathyroid hormone (PTH),glucagon-like peptide thymosin alpha I, IIb/IIIa inhibitor, alpha-1antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 inhibitors,bisphosphonates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin,aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate, polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V, penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate and where applicable, analogues,agonists, antagonists, inhibitors, and pharmaceutically acceptable saltforms of the above. In reference to peptides and proteins, the inventionis intended to encompass synthetic, native, glycosylated,unglycosylated, PEGylated forms, and biologically active fragments andanalogs thereof.

Thus, the invention includes a composition comprising a conjugate of apropionic acid-terminated polymer (or a reactive derivative thereof) anda biologically active molecule wherein the propionic acid-terminatedpolymer is prepared in accordance with the method described herein.Thus, for example, the conjugate in the composition can be formed byreacting

with an interferon, wherein m is defined as in Section II.A. Inaddition, the conjugate in the composition can be formed by reacting

with a TNFR tumor necrosis factor receptor, wherein m is defined as inSection II.A. Further, the conjugate in the composition can be formed byreacting

reacting with erythropoietin, wherein m is defined as in Section II.A.Also, the conjugate in the composition can be formed by reacting

with human growth hormone, wherein m is defined as in Section II.A.Variants and mimetics of the interferon, TNFR, erythropoietin, and humangrowth hormone can be substituted as the biologically active agent inthese conjugates. An exemplary conjugate will comprise a structurecorresponding to Formula IX:

wherein Active Agent represents a residue of an amine-containing activeagent and m is defined as in Section II.A.

Because the compositions include conjugates prepared from propionicacid-terminated polymers (or a reactive derivative thereof) formed bythe presently described method—which results in reduced degradation andchain cleavage of the polymer backbone—the resulting conjugates andcompositions likewise have reduced degradation and chain cleavage of thepolymer backbone, at least as compared to conjugates prepared frompropionic acid-terminated polymers (or reactive derivatives thereof)prepared by alternate methods (i.e., methods different from thepresently described method for synthesizing propionic acid-terminatedpolymers, or a reactive derivatives thereof).

In particular, the presently described method unexpectedly andadvantageously provides propionic-acid terminated polymers (as well asreactive derivatives and conjugates) that are more pure as a result ofreduced degradation and chain cleavage. The method particularly improvesthe purity when the propionic acid-terminated polymer comprisespropionic-acid terminated poly(ethylene oxide) bearing a methoxyend-cap.

For example, a propionic acid-terminated poly(ethylene oxide) bearing amethoxy end-cap [e.g., CH₃O—(CH₂CH₂O)_(m)—CH₂CH₂COOH)] comprises ethergroups, each ether group having an oxygen atom with a certainsensitivity to cleavage via nucleophilic attack. While not wishing to bebound by theory, it appears for at least three reasons that the mostsensitive ether group in the polymer prone to cleave is the ether groupassociated with the methoxy end-cap.

First, for reasons of accessibility, the ether group associated with themethoxy end-cap is the most exposed and subsequently relativelyavailable for a chain-cleaving nucleophilic attack. Second, the oxygenin the ether group associated with the methoxy end-cap is more basicthan the oxygens in the ether groups associated with the repeatingethylene oxide monomers. This is so because the oxygens in the ethergroups associated with the repeating ethylene oxide monomers have thebenefit of two neighboring ether oxygens (and their electron-withdrawingeffects) while the oxygen associated with the methoxy end-cap has only asingle neighboring ether oxygen. Third, while the oxygen in the ethergroup nearest the carboxylic acid group also only has a singleneighboring ether oxygen, this oxygen benefits from the neighboring andstability-enhancing carboxylic acid group that serves as a electronsink.

The above analysis can be illustrated in Schematic A, where the arrowsin the left structures show electron-donating and electron-withdrawingeffects of the attached groups and the right-hand structures arecompared for the relative stability of the protonated forms. InSchematic A, m is defined as in Section II.A.

Again, while not wishing to be bound by theory, it is believed that ofthe two right-hand structures in the above schematic, a nucleophile ismost likely to attack by backside nucleophilic displacement at thecarbon at the protonated methoxy oxygen (top, right-hand structure inthe above schematic). Reasons for this preference include a lower degreeof steric hindrance at the methyl group (thereby favoring methyl groupdisplacement) and a higher degree of steric hindrance within the polymerbackbone, as shown in Schematic B. In Schematic B, m is defined as inSection II.A.

Thus, for example, if a hydrogen sulfate anion is the nucleophile thatwill produce chain cleavage, the favored pathway for cleavage isbelieved to be the loss of the methyl group of the methoxy end-cap. SeeSchematic C, wherein m is defined as in Section ILA. This process willlead to formation of a hydroxyl end-cap in place of the methoxy end-cap(a “demethylated polymer”). This hydroxyl end-capped side productimpurity was neither observed nor reported in the “nitrile-based” methoddescribed in U.S. Pat. No. 5,672,662 because the hydroxyl end-cappedpolymer has essentially the same high performance liquid chromatography(“HPLC”) retention properties as the corresponding methoxy end-cappedspecies as each has essentially the same molecular weight. It will berecalled that the “nitrile-based” method described for preparingpropionic acid-terminated polymers in U.S. Pat. No. 5,672,662 requiresthe use of relatively harsh conditions such as one or more of (a) usingstrong concentrated mineral acids like sulfuric acid, or hydrochloricacid, (b) high temperatures, and (c) very long reaction times (exceeding30 hours in the case of hydrochloric acid-promoted hydrolysis).

It is preferred, then, that a composition disclosed in the invention issubstantially free of side product impurities bearing a hydroxyl end-cap(e.g., substantially free of HO—(CH₂CH₂O)_(m)—CH₂CH₂COOH species). Inthis regard, a composition that is substantially free of side productimpurities bearing a hydroxyl end-cap will contain less than about 15%by weight, more preferably less than about 10% by weight, morepreferably less than about 5% by weight, still more preferably less thanabout 3% by weight, yet still more preferably less than about 2% byweight of side product impurities bearing a hydroxyl end-cap, with lessthan about 1% by weight being most preferred.

The side product impurity—bearing a hydroxyl end-cap as show in FormulaX—can introduce additional unwanted species in a composition. Inparticular, a hydroxyl end-cap-containing impurity (such as that shownin Foimula X) can (i) compete with a reactive component of anothermolecule (e.g., the hydroxyl group of a reagent used to form polymerderivatives), and/or (ii) ultimately result in one or more hydroxylend-capped conjugate species.

The side product impurity (such as that shown in Formula X) can reactwith other molecules to form additional species that are not desired tobe present in the composition. For example, during esterification toform a reactive ester, the side product impurity bearing a hydroxylend-cap will compete with the hydroxyl group of the ester-formingN-hydroxysuccinimide reagent, thereby forming a dimeric species whereintwo polymers are linked. This unintended reaction does occur when, forexample, a side product impurity corresponding to Formula X is presentin the reaction mixture, thereby resulting in the formulation of thedimeric species shown in Formula XI:

wherein m is defined as in Section II.A. In contrast to a simplydemethylated impurity, the dimeric species is readily observable by gelpermeation chromatography (GPC) or HPLC because it has a highermolecular weight.

It is preferred, then, that a composition disclosed in the invention issubstantially free of species comprising a structure corresponding to adimeric species (e.g., hydroxyl end-capped and methoxy end-capped asshown in Formula XI). In this regard, a composition that issubstantially free of dimeric species will contain less than about 10%by weight, more preferably less than about 5% by weight, more preferablyless than about 4% by weight, still more preferably less than about 3%by weight, yet still more preferably less than about 2% by weight ofdimeric species, with less than about 1% by weight being most preferred.

As previously indicated, the hydroxyl end-cap-containing impurity (suchas the one shown in Formula X) can ultimately result in one or morespecies of hydroxyl end-capped polymer-active agent conjugates. Thus,for example, an impurity having a structure comprising a structurecorresponding to Formula X can—when subject to an esterificationreaction with N-hydroxysuccinimide—result in a species comprising astructure corresponding to Formula XII:

wherein m is defined as in Section II.A.

It should also be noted that a species comprising a structurecorresponding to Formula XII can result via an esterification reactionif the original starting methoxy end-capped poly(ethylene glycol)material used to prepare a propionic acid-terminated polymer iscontaminated with “diol” poly(ethylene glycol), i.e.,HO—(CH₂CH₂O)_(m)—H. In this regard, it is preferred to use startingmethoxy end-capped poly(ethylene glycol) compositions comprising lessthan 2% by weight of “diol” poly(ethylene glycol).

If a species comprising a structure corresponding to Formula XII issubsequently combined with an amine-containing active agent, then ahydroxyl end-capped conjugate having a species comprising a structurecorresponding to Formula XIII can be formed:

wherein Active Agent represents a residue of an amine-containing activeagent and m is defined as in Section II.A. A species comprising astructure corresponding to Formula XII could also lead to a conjugatecomprising a structure corresponding to Formula XIV:

wherein Active Agent represents a residue of an amine-containing activeagent and m is defined as in Section II.A. In addition, a speciescomprising a structure corresponding to Formula XII could result in aconjugate comprising a hydroxyl end-capped structure corresponding tocorresponding to Formula XV:

wherein Active Agent represents a residue of an amine-containing activeagent and m is defined as in Section II.A.

Each of the impurities comprising a structure corresponding to one ofFormulae X, XII, and XIII include a hydroxyl end-cap (or “demethylated”group). As pointed out above, the special concern with a compositioncomprising a relatively large amount of one or more of these or otherhydroxyl end-capped species is the ability of the impurity to react withan active agent when the composition is used in a conjugation reaction.The result can be the formation of second, third and fourth conjugatespecies, such as the intended conjugate (e.g, a species comprising astructure corresponding to Formula IX), and one or more unintendedconjugates (e.g., one or more species comprising a structurecorresponding to Formulas XIII or XIV. It has been found that mixturesof various conjugate species reduces the consistency, performance, andreproducibility of the resulting composition.

It is preferred that the composition is substantially free of conjugatespecies corresponding to any one or combination of Formulae XIII, XIV,and XV. In this regard, a composition that is substantially free ofconjugate species corresponding to any one or combination of FormulaeXIII, XIV and XV will contain less than about 10% by weight, morepreferably less than about 5% by weight, more preferably less than about4% by weight, still more preferably less than about 3% by weight, yetstill more preferably less than about 2% by weight of conjugate speciescorresponding to any one or combination of Formulae XIII, XIV, and XV,with less than about 1% by weight being most preferred.

EXPERIMENTAL

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention. For example, althoughmonofunctional PEG reagents are used in the examples to illustrate theinvention, difunctional or multifunctional PEG polymers could also beused in the present invention, as well as other types of water solubleand non-peptide polymers.

Unless otherwise noted, all PEG reagents referred to in the appendedexamples are available from Nektar AL of Huntsville, Ala. All NMR datawas generated by a 300 or 400 MHz NMR spectrometer manufactured byBruker.

Example 1 illustrates formation of a 20,000 Da mPEG-propionic acidpolymer using tert-butyl acrylate as the tertiary alkyl acrylate reagentand tetrabutylammonium hydroxide, formed in situ from tetrabutylammoniumbromide and potassium hydroxide, as the Michael addition catalyst. TFAis used to cleave the tert-butyl group. Example 2 is similar to Example1, except a 20,000 Da henzyloxy-PEG-propionic acid polymer is formedusing the direct addition of tetrabutylammonium hydroxide as thecatalyst. Example 3 is similar to Example 1, except the mPEG has amolecular weight of 30,000 Da.

Comparative Example 1 shows that the synthesis method outlined inExample 1 of U.S. Pat. No. 5,523,479 to Sanders et al. fails to producethe desired propionic acid, tert-butyl ester, when using a 20,000 Dapolymer starting material. It is believed that the method disclosed inU.S. Pat. No. 5,523,479 is ineffective in forming higher molecularweight propionic acid functionalized polymers of the type utilized inthe present invention. In particular, it is believed that the catalystssuggested in the Sanders patent are incapable of promoting the Michaeladdition reaction to any significant degree when a higher molecularweight polymer starting material is used.

Comparative Example 2 shows that the synthesis method outlined inExample 1 of U.S. Pat. No. 5,672,662 to Harris et al. fails to producepure m-PEG(20,000 Da)-propionic acid, when using a 20,000 Da low diolcontaminated methoxy-end-capped PEG starting material, because therelatively harsh reaction conditions lead to the demethylation and chaincleavage of methoxy-end-capped and produce substantial amount ofHO-PEG(20,000 Da)-propionic acid.

Example 1 Synthesis of mPEG(20,000 Da)-Propionic Acid

A. mPEG(20,000 Da)-Propionic Acid, tert-Butyl Ester

A solution of mPEG (20,000 Da) (35.0 g, 0.00175 moles) (NOF Corporation)and tetrabutylainmonium bromide (0.6 g) in toluene (125 ml) wasazeotropically dried by distilling off 105 ml of toluene. Potassiumhydroxide (0.15 g) in form of fine powder was added and the mixture waswarmed up to 60° C. under argon atmosphere. Then tert-butyl acrylate(2.0 ml, 0.01365 moles, 7.8 fold excess) was added during 2 h and themixture was stirred overnight at 60° C. under argon atmosphere. Next thesolvent was distilled off under reduced pressure and the residue wasdissolved in dichloromethane (400 ml). The resulting solution was washedtwo times with deionized water (2×50 ml) and then dried with anhydrousmagnesium sulfate. Next the solvent was distilled off under reducedpressure. Yield 28.5 g. NMR (d6-DMSO): 1.40 ppm (s, (CH₃)₃C—, 9H), 2.41ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH3, 3H), 3.51 ppm (s, PEGbackbone); substitution 69.1%.

B. mPEG(20,000 Da)-Propionic Acid

mPEG (20,000 Da)-Propionic Acid, tert-Butyl Ester (20g) from Step A wasdissolved in a dichloromethane/trifluoroacetic acid mixture (1:1; 120ml) and the solution was stirred 1 h at 60° C. After cooling to roomtemperature, dichloromethane (400 ml) was added to the reaction mixtureand the resulting solution was washed with deionized water (400 ml), anddried with anhydrous magnesium sulfate. Next the solvent was distilledoff under reduced pressure. The crude product was dissolved in deionizedwater (400 ml) and the pH of the solution was adjusted to 12 with 1.0 MNaOH. The solution was stirred 2 h at pH=12. Next NaCl (40 g) was addedand the pH was adjusted to 3 with 10-% phosphoric acid. The product wasextracted with dichloromethane, the solution was dried with anhydrousmagnesium sulfate, and the solvent was distilled off under reducedpressure giving 16.5 g of white solid product. Anion exchangechromatography showed that the product contains: m-PEG(20,000Da)-propionic acid 68.2% and m-PEG-20K 31.8%. Next the product waschromatographically purified using typical anion exchange chromatographymedia giving 100% pure PEG(20,000 Da)-propionic acid (9.8 g). In thisregard, “100% PEG(20,000 Da)-propionic acid” means 100% pure PEG(20,000Da)-monopropionic acid.

NMR (d₆-DMSO): 2.43 ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH₃, 3H),3.51 ppm (s, PEG backbone); No PEG-OH groups were detected by NMR (notriplet at 4.58 ppm); This experimental result means that the productwas 100% pure mPEG(20,000 Da)-monopropionic acid with no detectableHO-PEG(20,000 Da)-propionic acid present,

Example 2 Synthesis of Benzyloxy-PEG(20,000 Da)-Propionic Acid

A solution of benzyloxy-PEG (20,000 Da) (35.0 g, 0.00175 moles) (NOFCorporation) and tetrabutylammonium hydroxide (2.0 g of 40 wt % solutionin water) in toluene (200 ml) was azeotropically dried by distilling off175 ml toluene. The obtained solution was warmed up to 65° C. underargon atmosphere. Then tert-butyl acrylate (1.5 ml, 0.01024 moles, 5.85fold excess) was added during 3.5 hours and the mixture was stirredovernight at 60-65° C. under argon atmosphere. Next the solvent wasdistilled off under reduced pressure and the residue was dissolved indichloromethane (40 ml).

Trifluoroacetic acid (40 ml) was added and the solution was heated toboiling for 2 h. Dichloromethane and trifluoroacetic acid were distilledoff under reduced pressure and the crude product was dissolved in 400 mldeionized water. The pH was adjusted to 12 with 1.0M NaOH and thesolution was stirred 2 h at pH=12. Next NaC1 (40 g) was added and the pHwas adjusted to 3 with 10% phosphoric acid. The product was extractedwith dichloromethane, the extract was dried with anhydrous magnesiumsulfate, and the solvent was distilled off under reduced pressure giving28.5 g of white solid product. Anion exchange chromatography showed thatthe product contains: PEG(20,000 Da)-monopropionic acid 69.7% andPEG(20,000 Da) 30.3%. Next the product was chromatographically purifiedgiving 100% pure PEG(20,000 Da)-monopropionic acid. NMR (d₆-DMSO): 2.43ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEGbackbone), 4.49 ppm (s, —CH₂—, benzyloxy, 2H), 7.33 ppm (m, C₆H₅—, 5H).No PEG-OH groups were detected (no triplet at 4.58 ppm); This means thatthe product was 100% pure benzyloxy-PEG(20,000 Da)-propionic acid.

Example 3 Synthesis of mPEG(30,000 Da)-Propionic Acid

A. mPEG(30,000 Da)-Propionic Acid, tert-Butyl Ester

A solution of mPEG (30,000 Da) (50.0g, 0.00167 moles) (NOF Corporation)and tetrabutylammonium bromide (0.8 g) in toluene (200 ml) wasazeotropically dried by distilling off 100 ml toluene. Potassiumhydroxide (0.16 g) in form of fine powder was added and the mixture waswarmed up to 60° C. under argon atmosphere. Then tert-butyl acrylate(2.5 ml, 0.01707 moles, 10.2 fold excess) was added during 4h and themixture was stirred overnight at 60° C. under argon atmosphere. Next thesolvent was distilled off under reduced pressure and the residue wasdissolved in dichloromethane (400 ml). The obtained solution was washedtwo times with deionized water (2×100 ml) and then dried with anhydrousmagnesium sulfate. Next the solvent was distilled off under reducedpressure giving 42.5 g of solid product. NMR (d₆-DMSO): 1.40 ppm (s,(CH₃)₃C—, 9H) 2.41 ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51ppm (s, PEG backbone); substitution 73.8%.

B. mPEG(30,000 Da)-Propionic Acid

mPEG (30,000 Da)-Propionic Acid, tert-Butyl Ester (40 g) from Step A wasdissolved in a dichloromethane/trifluoroacetic acid mixture (1:1; 150ml) and the solution was stirred 1 h at 55° C. After cooling to roomtemperature, dichloromethane (600 ml) was added to the reaction mixtureand the solution was washed with deionized water (400 ml), and driedwith anhydrous magnesium sulfate. Next the solvent was distilled offunder reduced pressure. The crude product was dissolved in deionizedwater (800 ml) and the pH of the solution was adjusted to 12 with 1.0MNaOH. The solution was stirred 2 h at pH=12. Next, NaCl (80 g) was addedand the pH was readjusted to 3 with 10% phosphoric acid. The product wasextracted with dichloromethane giving 33.5 g of white solid product.Anion exchange chromatography showed that the product contains:PEG(30,000 Da)-monopropionic acid 67.7% and PEG(30,000 Da) 32.3%. Next,the product was chromatographically purified using typical anionexchange chromatography media giving 100% pure PEG(30,000Da)-monopropionic acid (25.3 g).

NMR (d₆-DMSO): 2.43 ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH₃, 3H),3.51 ppm (s, PEG backbone). No PEG-OH groups were detected (no tripletat 4.58 ppm); This means that the product was 100% pure mPEG(30,000Da)-propionic acid.

Comparative Example 1 Attempted Synthesis of mPEG(20,000 Da)-PropionicAcid, tert-Butyl Ester

Using the method outlined in Example 1 of U.S. Pat. No. 5,523,479, asolution of mPEG (20,000 Da) (35.0 g, 0.00175 moles) (NOF Corporation)in toluene (125 ml) was azeotropically dried by distilling off 105 ml ofsolvent. Potassium hydroxide (0.15 g) in the form of fine powder wasadded and the mixture was warmed to 60° C. under argon atmosphere. Thentert-butyl acrylatc (2.0 ml, 0.01365 moles, 7.8 fold excess) was addedduring 2 hours and the mixture was stirred overnight at 60° C. underargon atmosphere. Next, the solvent was distilled off under reducedpressure. Yield 36.5 g. NMR analysis showed that the polymer startingmaterial was unchanged: mPEG (20,000 Da). NMR (d₆-DMSO): 3.24 ppm (s,—OCH₃, 3H), 3.51 ppm (s, PEG backbone), 4.58 ppm (t, —OH, 1H).

Comparative Example 2 Synthesis of mPEG(20,000 Da)-Propionic Acidaccording to U.S. Pat. No. 5,672,662

A. mPEG(20,000 Da)-Propionitrile

A mixture of mPEG (20,000 Da) (25.0 g, 0.00125 moles) (NOF Corporation),distilled water (25.0 ml) and potassium hydroxide (0.5 g) was cooled to0-5° C. in an ice bath. Acrylonitrile (3.4 g) was added slowly, and thesolution was stirred for three hours at 0-5° C. Ten percent NaClsolution (225 ml) was added to the reaction mixture and the product wasextracted with dichloromethane (200, 100, and 50 ml). The organic layerwas dried over magnesium sulfate, and the solvent was distilled offunder reduced pressure. The crude product was dissolved indichloromethane (35 ml) and precipitated with isopropanol (225 ml) atroom temperature. The precipitate was removed by filtration and driedunder vacuum. Yield of M-PEG nitrile 23.5 g.

B. mPEG(20,000 Da)-Propionamide

A mixture of M-PEG nitrile from the above step (23.5 g) and concentratedhydrochloric acid (117.5 g) was stirred at, room temperature for 48hours. The solution was diluted with one liter of water and extractedwith dichloromethane (200, 150, and 100 ml). The combined organicextracts were washed twice with water, dried over magnesium sulfate,filtered, and concentrated to dryness by rotary evaporation. Yield ofPEG amide 21.5 g.

C. mPEG(20,000 Da)-Propionic Acid

M-PEG amide from the above step (16.0 g) was dissolved in 1150 ml ofdistilled water, 100 g of potassium hydroxide was added, and thesolution was stirred for 22 hours at room temperature. Sodium chloride(150 g) was added, and the solution was extracted with dichloromethane(150 ml×3). The combined organic extracts were washed with 5% phosphoricacid, water (twice), and dried over magnesium sulfate. Next, the solventwas distilled off under reduced pressure giving 14.0 g of white solidproduct. Anion exchange chromatography showed that the product contains:PEG(20,000 Da)-propionic acid 62.5%; and PEG-20K 37.5%. Next, theproduct was chromatographically purified using typical anion exchangechromatography media giving 100% pure PEG(20,000 Da)-monopropionic acid(6.5 g). NMR (d₆-DMSO): 2.43 ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s, —OCH₃,2.61 H), 3.51 ppm (s, PEG backbone, 1725 H), 4.58 ppm (t, PEG-OH, 0.13H). NMR analysis (triplet at 4.58) showed that the product contained 13mol % of PEG-OH groups; this means that the product was a mixture of thedesired mPEG(20,000 Da)-Propionic Acid (87%) andHO-PEG(20,000)-Propionic Acid (13%).

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included. Although specific termsare employed herein, they are used in a generic and descriptive senseonly and not for purposes of limitation.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

1. A composition comprised of a conjugate formed from the reaction of anactive ester of PEG and a biologically active molecule, wherein theactive ester of PEG is prepared by the method comprising: i) reacting aPEG polymer with a tertiary alkyl acrylate in the presence of a catalystcomprising a quaternary ammonium salt to form a propionic acid ester ofthe PEG polymer, wherein the PEG polymer comprises at least one hydroxylgroup and said PEG polymer is a monofunctional PEG, difunctional PEG, orbranched PEG molecule comprising 1 to about 25 hydroxyl groups andhaving a weight average molecular weight of at least about 10,000 Da;and ii) treating the propionic acid ester of the PEG polymer with astrong acid to form a PEG polymer functionalized with at least onepropionic acid group; iii) optionally, chromatographically purifying thePEG polymer functionalized with at least one propionic acid group; iv)derivatizing the PEG polymer functionalized with at least one propionicacid group to form an active ester of PEG; and v) optionally,chromatographically purifying the active ester of PEG.
 2. Thecomposition of claim 1, wherein the active ester is selected from thegroup consisting of N-succinimidyl ester, o-, m-, or p-nitrophenylester, 1-benzotriazolyl ester, imidazolyl ester, and N-sulfosuccinimidylester.
 3. The composition of claim 1, wherein the active ester isselected from the group consisting of:

wherein m is from 3 to 3000, and EC is a residue of a moiety selectedfrom the group consisting of fluorescein, biotin, acrylate,vinylsulfone, maleimide, tert-butyl carbonyl, and9-fluorenylmethoxycarbonyl.
 4. The composition of claim 3, wherein theactive ester comprises the structure

wherein m is 3 to
 3000. 5. The composition of claim 1, wherein thebiologically active molecule is selected from the group consisting of aninterferon, a TNFR, erythropoietin, human growth hormone, and variantsand mimetics of any of the foregoing.
 6. The composition of claim 1,wherein the tertiary alkyl acrylate is α- or β-substituted.
 7. Thecomposition of claim 6, wherein the substituting group is selected fromthe group consisting of halo, hydroxyl, thiol, alkylthio, acyl, acyloxy,nitro, cyano, azido, trihalomethyl, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl, andsubstituted heteroaryl.
 8. The composition of claim 7, wherein thesubstituting group is selected from the group consisting of methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,and benzyl.
 9. The composition of claim 1, wherein the tertiary alkylgroup of the tertiary alkyl acrylate is tert-butyl, tert-amyl,α,α′-dimethylbenzyl, trityl, 1-adamantyl, or 2-methyl-2-adamantyl. 10.The composition of claim 1, wherein the tertiary alkyl acrylate has thestructure:

wherein: R₁ and R₂ are each independently selected from the groupconsisting of hydrogen, halo, hydroxyl, thiol, alkylthio, acyl, acyloxy,nitro, cyano, azido, trihalomethyl, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heterocycle, substituted heterocycle, heteroaryl, andsubstituted heteroaryl; and R₃-R₅ are each independently alkyl,substituted alkyl, aryl or substituted aryl.
 11. The composition ofclaim 10, wherein R₃, R₄, and R₅, are each methyl, ethyl, or phenyl. 12.The composition of claim 10, wherein R₁ and R₂ are hydrogen.
 13. Thecomposition of claim 10, wherein R₁ is hydrogen and R₂ is selected fromthe group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, and benzyl,
 14. The composition ofclaim 1, wherein the strong acid is trifluoroacetic acid,trifluoromethancsulfonic acid, formic acid, hydrochloric acid, orp-toluenesulfonic acid.
 15. The composition of claim 1, wherein thecatalyst comprising the quaternary ammonium salt is selected from thegroup consisting of tetramethyl ammonium halide, tetraethyl ammoniumhalide, tetrapropyl ammonium halide, tetrabutyl ammonium halide,tetramethyl ammonium hydroxide, tetraethyl ammonium hydroxide,tetrapropyl ammonium hydroxide, and tetrabutyl ammonium hydroxide. 16.The composition of claim 1, wherein the catalyst comprising thequaternary ammonium salt has the structure:

wherein each R is independently alkyl or substituted alkyl and X is acounter ion.
 17. The composition of claim 16, wherein each R is C1-C8alkyl and X is halo or hydroxide.
 18. The composition of claim 1,wherein said reacting step and said treating step are conducted in thepresence of an organic solvent.
 19. The composition of claim 18, whereinthe organic solvent is selected from the group consisting ofdichloromethane, tetrahydrofuran, dimethylformamide, acetonitrile,toluene, xylene, phenyl acetonitrile, nitrobenzene, tetrachloroethylene,anisole, and chlorobenzene.
 20. The composition of claim 1, wherein theweight average molecular weight is from about 10,000 to about 100,000Da.
 21. The composition of claim 20, wherein the weight averagemolecular weight is from about 20,000 to about 60,000 Da,
 22. Thecomposition of claim 1, wherein the step of chromatographicallypurifying the PEG polymer functionalized with at least one propionicacid group is performed.
 23. The composition of claim 1, wherein thestep of chromatographically purifying the active ester of PEG isperformed.
 24. The composition of claim 1, wherein the step ofchromatographically purifying the PEG polymer functionalized with atleast one propionic acid group is performed and the step ofchromatographically purifying the active ester of PEG is performed.